A single layer of epithelial cells forms the barrier between the harsh environment of the gut lumen and the interstitium. In intestinal diseases such as inflammatory bowel disease, tight junctions, which seal the intercellular space, become leaky and the barrier is compromised. Inflammatory cytokines are known to contribute to this barrier defect, howevertheir mechanisms of action are poorly defined. The long term objectives of this application are to understand the unique regulatory mechanism that different cytokines have on barrier function. My central hypothesis, based on the preliminary data, is that different cytokines affect barrier function via functionally distinct mechanisms. I will test this hypothesis using standard tools as well as a novel approach I have developed that will allow high resolution analysis of tight junction biophysics. The predominant methods used in this study will allow biophysical characterization of the tight junction barrier and the effects of inflammatory cytokines. The first aim will characterize the distinct effectsof different cytokines, specifically TNF and IL13,on barrier function. Methods will include traditional current clamp and ion selectivity measurements coupled with macromolecular flux studies. These functional measurements will be correlated with measurements of protein transcription and expression. The regulatory mechanisms involved will be assessed through pharmacologic manipulation. The second aim will take advantage of and refine the approach I have developed to make high resolution biophysical measurements of barrier dynamics under normal conditions. These techniques will allow measurement of barrier function over extremely small areas on a millisecond time scale. The third aim will combine observations and techniques of the first two aims to determine the distinct effects of different cytokines on the biophysics of tight junction barrier function at high resolution. Completion of these aims will have significant positive effects on human health because it will greatly enhance our understanding of specific mechanisms by which cytokines cause barrier dysfunction. This will allow development of specific therapies targeting distinct pathways of barrier dysfunction. This holds great promise as we enter the era of individualized medicine, since availability of agents targeting different mechanisms of barrier dysfunction will allow treatment regimens designed for individual patients. Additionally, the techniques developed in the second aim will have utility in studying tight junction biophysics in other organs, such as kidney, lung, liver, and brain, where tight junction disruption is thought to contribute to disease.